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Abstract

Multi-scale multimodal microscopy is a very useful technique by providing multiple imaging contrasts with adjustable field of views and spatial resolutions. Here, we present a tri-modal microscope combining multiphoton microscopy (MPM), optical coherence microscopy (OCM) and optical coherence tomography (OCT) for subsurface visualization of biological tissues. The advantages of the tri-modal system are demonstrated on various biological samples. It enables the visualization of multiple intrinsic contrasts including scattering, two-photon excitation fluorescence (TPEF), and second harmonic generation (SHG). It also enables a rapid scanning over a large tissue area and a high resolution zoom-in for cellular-level structures on regions of interest. The tri-modal microscope can be important for label-free imaging to obtain a sufficient set of parameters for reliable sample analysis.

(a) MPM and (b) OCM images of 6 micron fluorescent microspheres. The overlay of MPM and OCM images is shown in Fig. 2(c) with color-coding of red and green respectively. Good co-registration is achieved with the overlapping of green and red to result in yellow. Scale bar represents 50 μm.

OCT, MPM and OCM images from leaf sample. Figure 3(a) shows the OCT cross-sectional image of the leaf; with the cuticle (C), lower epidermis (LE) and mesophyll spongy (MS) clearly visible. Dashed line shows the region where the MPM/OCM is performed. Figures 3(b)-3(c) show the MPM and OCM images of the leaf sample respectively. Structures visible are likely stomata (S) and papillae (P) of the cuticle. Figure 3(d) is the overlay of the (b) MPM and (c) OCM images with color codes of red and green respectively. Scale bars in Fig. 3(a) represent 100 μm; Figs. 3(b)-3(d) have the same scale with the scale bar in Fig. 3(b) representing 50 μm.

OCT, MPM, and OCM images from fish cornea sample. Figure 4(a) shows the large FOV cross-sectional OCT image where the epithelium (EP) and stroma (S) could be differentiated. Dashed line shows the region where the MPM/OCM is performed. Figures 4(b)-4(d) show the high-resolution TPEF, SHG and OCM images. Figure 4(e) shows the overlay of the TPEF, SHG and OCM images with color codes of red, blue, and green respectively. Scale bars in Figs. 4(a) and 4(b) represent 100 μm and 50 μm, respectively. Figures 4(b)-4(e) share the same FOV.

A stack of OCM images acquired together with the MPM images in Fig. 4 from the fish cornea sample. The frame numbers are labeled in the images. The OCM frame that co-registers with the MPM image in Fig. 4 is identified as frame number 256. The depth spacing between adjacent OCM frames is ~1 µm. The arrow shows the shifting direction of the boundary between the epithelium and stroma when the depth is increased. Scale bar is 50 µm.